Strain variation in Mycobacterium marinum fish isolates

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.     

Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis

Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.     

鲟分枝杆菌病及其病原研究

20092010年间,我国人工养殖的中华鲟（Acipenser sinensis）、史氏鲟（Acipenser schrencki）和杂交鲟（hybrid sturgeon:A. baeri-A. gueldenstaedtii）暴发了细菌性疾病。患病鲟通过组织切片观察,病原菌的分离、鉴定以及组织样品中病原菌的检测,结果显示从19条患病鲟中分离到49株分枝杆菌。病原菌经过多个保守基因的测序分析和部分生理生化特征的鉴定,共发现有7种分枝杆菌,分别为龟分枝杆菌（Mycobacterium chelonae）、海分枝杆菌（Mycobacterium marinum）、戈登氏分枝杆菌（Mycobacterium gordonae）、偶发分枝杆菌（Mycobacterium fortuitum）、苏尔加分枝杆菌（Mycobacterium szulgai）、猪分枝杆菌 （Mycobacterium porcinum）和Mycobacterium arpuense。在诊断过程中发现两种或三种分枝杆菌同时存在于同一样品中,分子生物学的诊断结果表明分枝杆菌复合感染十分常见,而海分枝杆菌是分枝杆菌复合感染中最为常见的分枝杆菌。分离的病原菌对斑马鱼的攻毒试验结果表明在以上7种分枝杆菌中海分枝杆菌的毒力最强。以上结果表明海分枝杆菌是鲟分枝杆菌病的主要致病菌,分枝杆菌复合感染是鲟分枝杆菌病的主要感染形式。研究中史氏鲟和中华鲟的分枝杆菌病,以及在病鱼体内分离的猪分枝杆菌和M. arupense在国内外均尚未见报道。       

Characterization of photochromogenic Mycobacterium spp. from Chesapeake Bay striped bass Morone saxatilis

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.     

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.     

Differentiation of Mycobacterium species by direct sequencing of amplified DNA

Nucleotide sequences specific for a range of Mycobacterium species were defined by computer-assisted sequence comparisons of small subunit ribosomal RNA. A polymerase chain reaction-based sequencing strategy was used to demonstrate that the 16S rRNA sequence can be used for the rapid identification of mycobacterial isolates. Identification at the species level can be obtained within 2 d, requiring less than 10,000 bacteria. This procedure reliably differentiates Mycobacterium spp. which are difficult to identify by classical methods, such as M. malmoense, M. szulgai and M. flavescens.     

Detection of mycobacteria in aquarium fish in Slovenia by culture and molecular methods

Thirty-five aquarium fish were investigated for the presence of mycobacteria by culture and molecular methods. The following species were examined: goldfish Carassius auratus auratus, guppy Poecilia reticulata, 4 three-spot gourami Trichogaster trichopterus, dwarf gourami Colisa lalia, Siamese fighting fish Betta splendens, freshwater angelfish Pterophyllum scalare, African cichlid fish Cichlidae spp., cichlid fish Microgeophagus altispinosus, cichlid fish Pseudotropheus lombardoi, blue streak hap Labidochromis caeruleus, sterlet Acipenser ruthenus, southern platyfish Xiphophorus maculatus, and catfish Corydoras spp. Isolates of mycobacteria were obtained in 29 cases (82.9%). Two specimens were positive using Ziehl-Neelsen (ZN) staining, but the cultivation failed. Four specimens were both ZN- and culture-negative. On the basis of GenoType Mycobacterium assay (Hain Life-science) and restriction enzyme analysis of the amplified products (PCR-RFLP), 23 isolates (79.3%) were identified: 7 as Mycobacterium fortuitum, 6 as M. gordonae, 6 as M. marinum, 3 as M. chelonae, and 1 as M. peregrinum. Five isolates remained unidentified (Mycobacterium spp.). One case probably represented a mixed infection (M. marinum/M. fortuitum). Since M. marinum infections are also detected in humans, the significance of mycobacteria in aquarium fish should not be overlooked.     

Isolation and characterization of mycobacteria from striped bass Morone saxatilis from the Chesapeake Bay

Mycobacteriosis in striped bass Morone saxatilis of Chesapeake Bay, USA, was first diagnosed in 1997 based on the presence of granulomatous inflammation and acid-fast bacteria in skin and spleen. To confirm histopathology, bacteriological detection and identification of mycobacteria were begun using splenic tissue from fish with and without skin ulcerations. On the basis of initial studies using a variety of selective and nonselective media, decontamination, homogenization and incubation conditions, a simple and quantitative recovery method using aseptic necropsy of splenic tissue was developed. Optimal recovery was obtained by spread-plating homogenates on Middlebrook 7H10 agar with incubation for 3 mo at 23 degrees C. Mycobacteria were recovered from 76% (n = 149/196) of fish examined. Mycobacterial densities exceeded 10(4) colony forming units x g tissue(-1) in 38% of samples (n = 63/168) that were examined using a quantitative approach. The most frequently recovered mycobacterium, present in 57% (n = 109/192) of characterized samples, was the recently named new species Mycobacterium shottsii. Polyinfections of M. shottsii and other mycobacteria were observed in 25% of samples (n = 47/192) with densities of M. shottsii usually 1 or more orders of magnitude higher than co-isolate(s). Other mycobacteria recovered included isolates that, based on phenotypic traits, resembled M. interjectum, M. marinum, M. scrofulaceum, M. szulgai and M. triplex. M. marinum, commonly associated with fish mycobacteriosis and human disease, was recovered infrequently (3%, n = 6/192). The presence of multiple mycobacterial types occurring at high densities suggests that a variety of mycobacteria could be causative agents of mycobacteriosis in striped bass from the Chesapeake Bay. Striped bass is the major recreational fish species in the Chesapeake Bay, and the significance of the current epizootic to human health and the potential adverse effects on fish stocks are not known.     

Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae

The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T.?soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T.?soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1?pg DNA per reaction (相似文献   

First report of a mycolactone-producing Mycobacterium infection in fish agriculture in Belgium

In the past few years, a mycolactone-producing subgroup of the Mycobacterium marinum complex has been identified and analyzed. These IS 2404 -positive species cause pathology in frogs and fish. A recently isolated mycobacterial strain from a fish in Belgium was analyzed using a variety of molecular methods and the results were identical to those obtained from a mycolactone-producing M. marinum from Israel.     

Detection and identification of mycobacteria by amplification of mycobacterial DNA

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.     

Use of a novel multiplex probe array for rapid identification of <Emphasis Type="Italic">Mycobacterium</Emphasis> species from clinical isolates

Conventional identification of mycobacteria is based on the analysis of their phenotypic and biochemical characteristics after culture; thus this method is time-consuming, laborious, and is not always conclusive. Developing a fast and accurate method for rapid identification of Mycobacterium species is in urgent need for early diagnosis of mycobacteriosis and effective patient management. In this study, an efficient and affordable novel multiplex probe array which allows simultaneous identification of 15 medically important mycobacterial species was developed. A pair of genus-specific primers and a set of genus- and species-specific probes were designed according to the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer (ITS) sequence, and 23S rRNA gene of mycobacteria. This probe array was applied for the identification of 78 clinical mycobacterial isolates recovered from Henan, China. The results showed that the specificity and sensitivity of the probe array were 100% for both genus-specific probe and Mycobacterium tuberculosis complex-specific probe. Among 52 isolates of nontuberculous mycobacteria, 43 isolates (82.7%) can be rapidly identified to the species level. Genetic variability of 16S-23S rRNA gene ITS region in M. avium, M. intracellulare, M. chelonae, M. abscessus and M. fortuitum were analyzed. With the accumulation of the sequences of ITS identified and further optimization of probes, the multiplex probe array has the potential to be developed into a practical tool for rapid and accurate identification of mycobacterial species in clinical laboratory.     

Chronic Mycobacterium marinum infection acts as a tumor promoter in Japanese Medaka (Oryzias latipes)

An accumulating body of research indicates there is an increased cancer risk associated with chronic infections. The genus Mycobacterium contains a number of species, including M. tuberculosis, which mount chronic infections and have been implicated in higher cancer risk. Several non-tuberculosis mycobacterial species, including M. marinum, are known to cause chronic infections in fish and like human tuberculosis, often go undetected. The elevated carcinogenic potential for fish colonies infected with Mycobacterium spp. could have far reaching implications because fish models are widely used to study human diseases. Japanese medaka (Oryzias latipes) is an established laboratory fish model for toxicology, mutagenesis, and carcinogenesis; and produces a chronic tuberculosis-like disease when infected by M. marinum. We examined the role that chronic mycobacterial infections play in cancer risk for medaka. Experimental M. marinum infections of medaka alone did not increase the mutational loads or proliferative lesion incidence in all tissues examined. However, we showed that chronic M. marinum infections increased hepatocellular proliferative lesions in fish also exposed to low doses of the mutagen benzo[a]pyrene. These results indicate that chronic mycobacterial infections of medaka are acting as tumor promoters and thereby suggest increased human risks for cancer promotion in human populations burdened with chronic tuberculosis infections.     

应用基因芯片快速检测分枝杆菌

目的:将临床疑似分枝杆菌感染患者的标本用基因芯片方法进行分枝杆菌菌种鉴定,并将基因芯片检测的结果与传统的抗酸染色方法进行对比。方法:采用基因芯片PCR扩增、分子杂交、微阵列芯片扫描的方法检测379例临床疑似标本。结果:基因芯片阳性检出率为16.3%(62/379),涂片抗酸染色法阳性检出率为15.3%(58/379),两者差异无统计学意义(χ2=0.16,P>0.05);62例基因芯片检测阳性患者中有52例为结核分枝杆菌,另有10例为非结核分枝杆菌(其中3例偶然分枝杆菌,1例龟/脓肿分枝杆菌,1例堪萨斯分枝杆菌,4例浅黄分枝杆菌,1例海分枝杆菌)。结论:结合临床病例分析结果显示,基因芯片检测对鉴别结核分枝杆菌和非结核分枝杆菌分型具有快速、特异性高的特点,在分枝杆菌感染的早期诊断和治疗上具有重要的临床价值,值得推广和应用。     

Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria

Aims: To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S–23S rRNA internal transcribed spacer (ITS) region for timely patient management. Methods and Results: Two mPCR assays were developed: Slow‐Growers (SG) mPCR was used for the detection of slow‐growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast‐Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species‐specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S–23S ITS region and when further confirmation was needed, hsp65 sequencing was performed. Conclusions: The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM. Significance and Impact of the Study: In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005–2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.     

Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units

A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n = 39), M. marinum (n = 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Boreal soils have been suspected reservoirs of infectious environmental mycobacteria. Detection of these bacteria in the environment is hampered by their slow growth. We applied a quantitative sandwich hybridization approach for direct detection of mycobacterial 16S rRNA in soil without a nucleic acid amplification step. The numbers of mycobacterial 16S rRNA molecules found in the soil indicated the presence of up to 10(7) to 10(8) mycobacterial cells per gram of soil. These numbers exceed by factor of 10 to 100 x the previous estimates of mycobacteria in soil based on culture methods. When real-time PCR with mycobacteria targeting primers was used to estimate the number of 16S rDNA copies in soil, one copy of 16S rDNA was detected per 10(4) copies of 16S rRNA. This is close to the number of 16S rRNA molecules detected per cell by the same method in laboratory pure cultures of M. chlorophenolicum. Therefore a major part of the mycobacterial DNA in the studied soils may thus have represented metabolically active cells. The 16S rRNA sandwich hybridization method described in this paper offers a culture independent solution for tracking environmental reservoirs of viable and potentially infectious mycobacteria.     

Pathogenesis of Mycobacterium spp. in zebrafish (Danio rerio) from research facilities

One of the most common diseases that we have diagnosed in zebrafish is mycobacteriosis, caused by several Mycobacterium spp. The severity of the disease ranged from severe outbreaks to incidental infections. We conducted an in vivo study to evaluate the pathogenesis of six isolates of Mycobacterium from zebrafish with mycobacteriosis from four research facilities and one wholesale supplier of zebrafish in the United States: Mycobacterium abscessus, Mycobacterium peregrinum, Mycobacterium chelonae (2 isolates), and Mycobacterium marinum. We also included two isolates of M. marinum from other fishes. Fish were exposed by intraperitoneal injection at a target does of 5 x 10(4) bacteria/fish, and were held in static aquaria at 28 degrees C for 8 weeks. Fish were examined by histology and culture, and mortalities were recorded. The M. marinum isolates caused 100% infection and mortality between 30% and 100%. None of the other Mycobacterium species caused significant mortalities, but several of these fish had granulomatous lesions in visceral organs. Mycobacteria were consistently recovered in culture from fish exposed to M. marinum, and from only 9% of fish exposed to the other species. This study suggests that, of the isolates tested, only M. marinum is highly pathogenic and virulent to healthy zebrafish.     

Conserved mechanisms of Mycobacterium marinum pathogenesis within the environmental amoeba Acanthamoeba castellanii

Mycobacterium marinum is a waterborne mycobacterial pathogen. Due to their common niche, protozoa likely represent natural hosts for M. marinum. We demonstrate that the ESX-1 secretion system is required for M. marinum pathogenesis and that M. marinum utilizes actin-based motility in amoebae. Therefore, at least two virulence pathways used by M. marinum in macrophages are conserved during M. marinum infection of amoebae.